The global agriculture industry faces many challenges and pressures that are particularly evident in the production of sessile organisms: biotic and abiotic stresses threatening crop yield and quality; increasing labour, water and energy costs; and further constraints imposed by consumer preference. As such, there is great demand to produce crops that are stress tolerant, require little or no input (i.e. reduced use of water, fertilizer, and/or pesticides), and are also appealing to consumers. The possibilities for trait development using traditional breeding are becoming increasingly limited due to a lack of genetic diversity in cultivated plant varieties. Introgression of valuable traits from wild accessions is possible, but this approach might not be feasible if the trait of interest is closely linked to those associated with undesirable traits (Fitzpatrick et al, Plant Cell. 24:395-414). A transgenic approach can be pursued, but genetically-modified organisms, particularly those yielding edible products, are controversial and present entirely new challenges with respect to food safety regulations and consumer acceptance. Mutagenesis is an effective and efficient method to introduce genetic diversity in crop plants (Wang et al, Plant Biotechnology Journal 10:761-772).
Mutagenesis of plants for crop improvement is a technique that has been used for nearly a century. Some of the first reports of human-directed plant mutagenesis come from Lewis Stadler who, in the 1920′s, irradiated barley and maize with X-rays and ultraviolet light (Stadler 1932). Chemical mutagenesis was introduced as an alternate technique making use of alkylating reagents such as ethylmethane suphonate (EMS), which disrupts DNA and leads to heritable changes in the genome (Koornneef et al. 1982). There have been many successes in using mutagenesis to develop new traits and new varieties of crops. The International Atomic Energy Agency (IAEA) which is a branch of the United Nations Food and Agriculture Organization (FAO) headquartered in Vienna, Austria provide a database of registered varieties derived from mutagenesis (mvga.isea.org). A popular example of a mutant variety is Ruby Red Grape fruit, released in 1970 in Texas, which used irradiation to improve the red colour and colour retention of grapefruit. Also, Golden Promise barley was produced through mutation causing semi-dwarfism, higher yield and salt tolerance (Forster 2001). Both of these varieties have been used extensively to produce modern day varieties of grapefruit and barley.
Alternatives to mutagenesis for inducing changes to the genome have been developed in the last few decades. Methods include using artificial constructs, often hybrids of DNA/RNA with other amino acids or nucleic acids (Sargent et al, Oligonucleotides 21(2):55-75, 2011, KEYBASE® by KeyGenex), to induce genetic changes at a specific location(s). The hybrid molecules are delivered into the cell through biolistics or membrane fusion where they then migrate to the nucleus to affect a change to the genome. The affected cells are then coaxed to grow into full plants. More recently site specific mutagenesis through the use of designer enzyme systems have been developed. Zinc-finger nucleases, transcription activator-like effector nucleases and meganucleases are all specifically designed enzymes that have the ability to cut genomic DNA at a desired location. These enzymes are either expressed in the cell(s) of a plant from a transgene construction introduced to the genome or from an extra-genomic fragment present within the cell. Recently they have been delivered into plant cells as proteins, eliminating the need of introducing foreign DNA into the plant cell. All of these enzymes are able to cut the double-strand DNA of the genome. This activates the cell's DNA repair mechanism which allows the incorporation of a new fragment of DNA through homologous recombination, or the plant closes the break, inducing some kind of change, be it an insertion, deletion or substitution through the process of non-homologous end-joining. More recently a new type of designer nuclease has been developed based on the bacterial immunity mechanism called the CRISPR/Cas system. The main benefit of the CRISPR/Cas system is that an RNA molecule is used to give the complex its genome editing specificity. This is in contrast to all other previous nuclease technologies where genome specificity was achieved by designing custom DNA binding protein domains. With CRISPR/Cas, genome targeting occurs through the complementary pairing of an RNA guide molecule to a target region, so customizing a new nuclease is as easy as designing a new RNA guide sequence. Like previous nucleases, the constructs expressing this system are placed into a cell where it is expressed and modifies the genome.
A genetic screen is used to identify and select for individuals who possess a phenotype of interest in a mutagenized or modified population. Types of genetic screens include forward genetic screens and reverse genetic screens. A forward genetic screen first identifies individuals of a mutant population with a particular phenotype of interest. Once the phenotype has been found, follow-on work is done to identify the underlying genetic change that led to the creation of the new trait. For example, Nordstrom et al. (Nat. Biotech. 31(4):325-331; 2013) utilize k-mers (short, overlapping nucleic acid fragments) in a forward genetic screen to identify mutations by direct comparison of whole-genome sequencing data from mutant and wild-type individuals. A reverse genetic screen, opposite to a forward genetics screen, analyzes the phenotype of an organism following the disruption of a known gene.
Mutagenesis of crop plants was, until recently, exclusively used in a forward genetic screen approach, whereby large mutagenized populations were phenotyped in search of novel traits for crop improvement. This approach is labour intensive, prone to identifying false positives and constrained by what can be measured visually or by high throughput methods.
Because of the labour associated with forward genetic screens, mutagenesis and mutation breeding lost popularity until the prospects of applying molecular biology were considered.
Targeting induced local lesions in genomes (TILLING) is a reverse genetics approach whereby high density, random mutagenesis of populations and single nucleotide polymorphism (SNP) detection techniques are combined to identify plants with valuable mutations (McCallum et al. 2000). High-Resolution DNA Melting (HRM) has been used in TILLING approaches for mutation detection in EMS-treated populations (Gady et al, Plant Methods 5:13), however this approach is labour intensive and expensive.
SNP detection in plants has quickly advanced since 2000 and now it is common to use next generation sequencing (NGS) to discover SNPs in populations of plants (Missirian et al. 2011, Rigola et al. 2009, Tsai et al., 2011).
Next generation DNA sequencing (NGS) is an appealing tool to identify mutations in populations of individuals. The rapidly falling price, ever increasing throughput and complete DNA characterization of the sequencing targets has drawn researchers to investigate NGS as a TILLING tool (Rigola et al, PLoS One 4:e4761; Tsai et al, Plant Pysiology 156:1257-1268). However, due to the intrinsic error-rate of NGS technologies it is difficult to discern mutation from sequencing mistakes in pools of thousands of individuals. Illumina sequencing technology produces a base-calling error almost twice every 1000 bases sequenced (Minoche et al, Genome Biology 12:R112). In an effort to differentiate errors from mutation, researchers have created multi-dimensional pooling strategies combined with DNA barcoding to sequence members of a population in multiple, independent reactions. Individuals harbouring a mutation are then determined by pool deconvolution using the barcodes (Rigola et al, PLoS One 4:e4761; Missirian et al, BMC Bioinformatics 12:287; WO2007037678 to KeyGene N.V.). Strategies which utilize composite reads to reduce error rate have also been developed (WO2014/134729).
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.